Apparatus for the cultivation of anaerobic and microaerophilic organisms



APPARATUS FOR CULTIVATION OF ANAEROBIC AND MICRO-AEROPHYLIC ORGANISMS Filed Feb. 16, 1942 .May 9, 1944- J H. V,BRI-:WER 2,348,448

John H Lewe,

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Patented May 9, 1944 ANAEROBIC AND GANISMS MICROAEROPIIILIC OR- John H. Brewer, Towson, Md., assignor to Kimble Glass Company, Vineland, N. J., a corporation of Illinois Application February 16, 1942, serial No. 431,179

s claims. (o1. iss-139) The present invention relates to apparatus for the cultivation of anaerobic and micro-aerophilic organisms. Specifically the invention pertains to a `modified cover for culture dishes ordinarily employed in the process of growing bacteria, commonly known as Petri dishes.

l In the cultivationof certain organisms, it has been established that some grow in the `presence ,of air or free oxygen, and are known as aerobes,

whileothers thrive more readily in the absence of freepXygen, and are generally referred to as anaerobic organisms. Micro-aerophils are organisms which require a reduced oxygen tension.

. 4Various forms of apparatus have been devised to reduce the free oxygen content in or surrounding the medium which supports anaerobic'organisms for cultivation therein, but they are bulky and, in most instances, too costly for ordinary laboratory which is only slightly less than that of the web portion so that aiiat, smooth annular face or rim I5 lying wholly in one plane is juxtaposed to and surrounds the recess. The lower terminal edge I6 of outer skirt I1 of the U-shaped flange I3 extends somewhat below face I5 so that, when the cover is resting on a flat surface I8 as shown in Fig.'3, lface I5 is spaced from and out of contact with said surface. 'I'his prevents contamination of face I5 by matter on the supporting table and likewise prevents matter on the face from coming in contact with the table surface to contaminate the latter.

As shown in Figf. the Petri dish is partially lled with medium I9 consisting of a gel-like substance containing an infusion broth, a gel or mucoid, and an agent forreducing the free oxyuse. Nor are the culture dishes employed in such apparatus readily accessible for removal or observation.

It is an object of the present invention to provide a novel cover for Petri dishes which is not only inexpensive but which is so designed that it permits cultivation of organisms without the employment of other apparatus or the use of oxygen reducing agents other than those incorporated in the medium supporting the organisms.

Other obj ects of the invention will be apparent from the following description of the invention taken in connection with the accompanying drawing, in which I i Fig. 1 is a perspective of'a Petri dish and cover therefor, the cover being removed from the dish and illustrating a preferred form of the invention;

Fig. 2 is a section taken through the dish and cover, the dish containing an organism supporting medium upon which the cover rests and forms an airtight seal for the culture; and

Fig. 3 illustrates the manner in which the cover may rest on a supporting surface such as a table.

In the drawing, the conventional circular glass Petri dish is indicated at I0, and, as shown in Fig. 1, has an annular vertical wall II. The cover, which constitutes the novel feature of the apparatus, is molded or otherwise formed from glass or other suitable plastic material and comprises a circular plate or web portion I2 having an annular inverted- U-shaped flange I3 therearound. It will be seen that this ange bridges the side wall II. The web portion I2 has substantial thickness, but is thin enough to permit observation therethrough and its lower face is provided with a shallow circular recess I4, the diameter of cator.

gen. Other materials may be added to the medium to support the growth of the organisms if desired, as well as to provide a reduction incli- A suitable formula for the medium includes a meat infusion, sodium thioglycollate, peptone, agar and dextrose, all incorporated in an aqueous solution and permitted to set. Silica gel or gelatine may be used instead of agar and in place of sodium thioglycollate there may be used'any other satisfactory reducing agent. The

agar or similar gel provides little or no nutriment,

but is combined with the other ingredients toform a supporting medium for the organisms and to permit surface cultivation thereon. Methylene blue in low concentration may also be added as an I indicator, the medium then remaining'a ,bluegreen while oxygen is present, but becoming clear when the medium is anaerobic, i. e., when the oxygen has been reduced.

When the medium has set and the culture deposited on the surface thereof, the cover 'is placed over the dish in such a way that the annular face I5 rests on the surface' of the medium, the annular side wall II being disposed in groove 20 of the U-shaped ange. It is to be noted that thc height of the medium in the dish is such that the top edge of the side wall II is spaced from the bend in flange I3, the cover being supportedsolely by the medium I9. This sistency that the glass surface of face I5 forms a seal therewith. In this way a small amount of air is trapped in recess I4 and other air is prevented from entering the recess. As compared with the depth of dish I0, which might be 10 mm. or 15 mm., the depth of recess I4 is only 1 mm., so that the amount of air trapped therein over the surface of the medium is obviously small. The reducing agent in the medium absorbs the oxygen medium is of such a con-V anaerobic condition exists.

-ameter of web I2. recess I4 has a definite relationto the diameter of the bottom of the culture dish-i.

` -of the dish is twice that f the recess.

in this small amount ofI air and thereafter an It is to be emphasized thatno additional sealing medium, such as petrolatum, parain, or

molding clay commonly used in sealing culturey dishes, is necessary to effect an air-tight seal for the shallow chamber in which the culture is grown. 'Ihe growth of the organisms is promoted on the surface of the medium so that,

, when the cover is removed, they may easily be inspected or, if desired, transplanted. The mediuin being relatively clear .and the dish and cover transparent, it is obvious that the dish may be removed from the incubator for examination in transmitted light at any time, without contaiminating the dish or necessitating breaking of the seal. It is also apparent that use of the comparativelyinexpensive dish and novel cover eliminates the necessity for employing any other chemical or physical agent to produce anaerobiosis other than that contained in the medium itself. Furthermore, by observing moderately careful bacteriological technique, the plates may be handled in the same manner as the conventionalculture dish without danger of contaminating the surface of the medium with agents used as reductants and, for this reason, the dish may be incubated either in the position shown in Fig. 2 of the drawing or in an inverted position.

It has been stated hereinbefore that the diameter of recess I4 is somewhat less than the di- Preferably the diameter of This relation should be such that the area of the bottom With such an arrangement if one counts the bacterial colonies developing un'der the recess of the dish and multiplies by two it is possible to make a plate count of anaerobes using this dish. In making such a count it would be necessary to make a pour plate rather than a streak plate, a pour plate being made as follows: 1 cc. or a fraction thereof of bacterial culture containing an unknown number of organisms is placed in the melted agar and mixed before it is solidified. Then when these bacteria develop sufliciently to permit the colonies to become visible, it is possible to count the colonies and ascertain the number of bacteria in the original culture.

When removing the cover from the dish, the

ble or the sealing face of the cover.

While the dish and cover and portions thereo have been described as beingcircular or annular, it is obvious that they may assume other shapes in transverse cross section without departing from the principles set forth' herein and as delined in the appended claims. For this reason,

the invention Ais not limited to dishes or covers which are circular. l

As explained hereinbefore, the removed Having fully descibed my invention, what I vclaim as new and desire to secure by Letters Patent is:

1. A cup-shaped cover rfor Petri dishes comprising a substantially flat transparent plate having a relatively shallow recess in the underside thereof, said underside having a smooth flat rim portion juxtaposed to and surrounding said recess, and an upstanding peripheral flange on said plate.

2. A cup-shaped cover for Petri dishes comprising a substantially flat transparent plate havving a relatively shallow recess in the underside thereof, said underside having a smooth iiat rim portion juxtaposed to and surrounding said recess, and an upstanding peripheral U-shaped ange onsaid'plate, the outer portion of said aglge comprising a skirt spaced laterally from said rim portion, the edge of said skirt lying in a plane parallel to and below that of said ilat rim portion.

3. In combination with a Petri dish adapted to contain a gel-like culture supporting medium, a cover for the dish comprising a. substantially at transparent plate having a relatively shallow recess in one face thereof, said face having a smooth flat rim portion juxtaposed to and surrounding said recess, said rim portion being adapted to rest on the contained gel-like medium to form an air-tight seal therewith.

4. In combination with a Petri dish adapted to contain a gel-like culture supporting medium, a cover for the dish comprising a substantially at transparent plate having a relatively shal-v low recess in one face thereof, said face having a smooth flat rim portion juxtaposed to and surrounding said recess, said rim portion being adapted to rest on the contained gel-like medium to form an air-tight seal therewith, and an inverted U-shaped flange on said plate, said flange bridging the side wall of said dish.

5. In combination with a Petri dish adapted to contain a gel-like culture supporting medium y containing anoxygen reducing agent, a cover for flat transparent plate having a relatively shallow the dish, said dish being recessed in one face thereof and having a smooth rim portion juxtaposed to'andsurrounding said recess, said rim portion being adapted to rest on the contained gel-like medium to form an air tight seal therewith, said recess being of such capacity' that a predetermined volume of air is trapped adjacent the surface of the medium, the oxygen in said trapped air being substantially completely reduced by the reducing agent in said medium.

6. In combination with a Petri dish adapted to contain a gel-like culture supporting medium, a cover for the dish comprising a substantially recess in one face thereof, said face having a smooth flat rim portion juxtaposed to and surrounding said recess, said rim portion being adapted to rest on the contained gel-like medium to form an air-tight seal therewith, the transverse cross-sectional area of the Petri dish being twice that of the transverse cross-sectional area of said recess.

.JOHN H. BREWER. 

